The ratiometric fluorescence nanoparticle based on SiRB for pH detection of tumor

Article abstract of DOI:10.1016/j.ejps.2018.03.015

Tumor pH detection and pH value change monitoring have been of great interest in the field of nanomedicine. In this study, a pH-sensitive near-infrared fluorescence probe SiRB (Si-rhodamine and Boronic acid group) was synthesized by introducing a boronic

Full text of DOI:10.1016/j.ejps.2018.03.015

EuropeanJournalofPharmaceuticalSciences118(2018)32–39
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
The ratiometric fluorescence nanoparticle based on SiRB for pH detection of
tumor
⁎
Qinghui Wanga,1, Xiaoyu Dinga,1, Yuhui Wangd, Qian Dua, Tianguo Xua, Bin Dua,b,c,
,
⁎
Hanchun Yaoa,b,c,
a
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, China
Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province, Zhengzhou 450001, China
The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450001, China
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tumor pH detection and pH value change monitoring have been of great interest in the field of nanomedicine. In
this study, a pH-sensitive near-infrared fluorescence probe SiRB (Si-rhodamine and Boronic acid group) was
synthesized by introducing a boronic acid group into the silicon rhodamine structure. ICG (Indocyanine green) as
the fluorescence internal standard and SiRB were loaded into PLGA (poly lactic-co-glycolic acid) to form PLGA-
SiRB-ICG nanoparticle. The experiments showed that the size of the nanoparticle was about 90 nm, which can
reach tumor passively by enhancing permeability and retention effect. PLGA in the acidic environment will
accelerate the release of cleavage, and the fluorescence ratio of the two probes can reflect the specific pH value
in the tumor. The results indicated that the nanoparticle could quantitatively measure the pH value of the tumor
site, which is expected to be used in tumor research and treatment.
Tumor microenvironment
Fluorescence imaging
pH detection
Fluorescence switch
1. Introduction
the ratiometric imaging of lysosomal HClO in living cells; Chen et al.
designed a fluorescent sensor for sensitive and selective detection of
Tumor microenvironment is a complex internal environment com-
posed of tumor cells, interstitial cells and extracellular matrix
(Gandellini et al., 2015; Hui and Chen, 2015; Wu and Dai, 2017;
Alkatout et al., 2017). The characteristics of tumor microenvironment,
such as high interstitial fluid pressure, hypoxia, and low extracellular
pH, are closely related to the various prognostic factors that control the
tumor growth and metastasis (Yang and Yu, 2015; Joyce, 2005). Studies
have shown that the tumor acidic environment is caused by anaerobic
glycolysis and acidic substance removal disorder (Zhao et al., 2016).
Not only would it increase the potential of tumor migration and inva-
sion, but also obstruct the activities of p53 and p-glycoprotein, leading
to the MDR (multidrug resistance) in chemotherapy (Florence et al.,
2015; Yan and Jurasz, 2016). Therefore, developing a rapid and accu-
rate measurement method of tumor microenvironment pH is conducive
to the diagnosis and prognosis of the tumor.
copper(II) ion and sulfide anion (Wang et al., 2017; Chen et al., 2016).
Among the methods of detecting tumor pH, one way is to connect the
fluorophore to the quencher via the hydrazone bonds, which would
break in acidic environment to control the fluorescent switch (Li et al.,
2017). The drawback of this way is that the hydrazone bonds may
rupture during the blood circulation and cause the fluorescence emis-
sion before reaching the tumor site. There is also a strategy is adding
electron-withdrawing groups to the fluorescent groups, to control the
fluorescence emission by PET (photoelectron transfer effect) (Lau et al.,
2014). The disadvantages of this strategy are that the sensitivity of the
fluorescent switch is low and the electron-withdrawing groups cannot
completely inhibit the emission of fluorescence. Moreover, these
methods can only compare the level of tumor pH, but cannot measure
the specific value of pH. So it is urgently needed to develop an accurate
and easy-to-operate method for pH imaging.
With the development of fluorescent imaging technology and its
application in medical imaging, more and more fluorescent probes are
synthesized for the detection of small molecule substances. For in-
stance, Wang et al. reported a FRET-based fluorescent polymer dots for
As a novel silicon-containing rhodamine derivatives, SiR (Si-rho-
damine) has received extensive attention recently. The reason is that
SiR has not only excellent photophysical properties and biocompatible
properties, but also the excitation and emission wavelengths in the
⁎
Corresponding authors.
E-mail addresses: dubinpaper@sina.com (B. Du), yhchpaper@sina.com (H. Yao).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejps.2018.03.015
Received 22 November 2017; Received in revised form 6 March 2018; Accepted 14 March 2018
Availableonline15March2018
0928-0987/©2018PublishedbyElsevierB.V.

Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
near-infrared region (Tao et al., 2017; Zhu et al., 2015). So it is parti-
cularly suitable for tumor tissue imaging. What is more, the spir-
ocyclization of SiR is an attractive strategy for designing NIR (near
infrared) fluorescent probes to detect some ions by a reversible ring-
opening process (Zhu et al., 2015; Wang et al., 2012). Following this
design principle, some SiR-based fluorescent dyes have been exploited
for imaging Hg2+, OH−1 and the proteins of interest from living cells.
As a promising recognition group, boronic acid participates in such an
equilibria: in a neutral or mildly basic environment, it binds with the
substances containing hydroxyl groups and forms the corresponding
boronate which can be reversibly hydrolyzed to boronic acid under
acidic conditions (Dervisevic et al., 2017; H.S et al., 2017). Herein we
designed and synthesized a SiR-based spirocyclic derivative SiRB (Si-
rhodamine and boronic acid), in which a boronic acid group was in-
anhydrous Na2SO4. After removal of the solvent under reduced
pressure, the residue was purified by silica gel column
chromatography with petroleum ether/dichloromethane (2/1, v/v) as
eluent. The pure compound 1 was a white solid (yield: 71%).
2.2.1.2. Synthesis of SiX (Si-xanthone). The compound 1 (5 mmol) and
anhydrous THF (tetrahydrofuran) (20 mL) were added to a pre-dried
flask flushed with nitrogen. The solution was cooled to −78 °C, 1.3 M s-
BuLi (sec-butyllithium) (10.7 mL) was added, and the mixture was
stirred for 30 min. At the same temperature, a solution of SiMe2Cl2
(10 mmol) in anhydrous THF (10 mL) was slowly added, and the
mixture was slowly warmed to room temperature, then stirred for
3 h. The reaction was quenched by the addition of 2 M hydrochloric
acid. Then the mixture was neutralized with NaHCO3 solution, and
extracted with CH2Cl2. The organic layer was washed with brine and
dried over anhydrous Na2SO4. The solvent was removed under reduced
pressure and the crude was used without further purification. KMnO4
solution (15 mmol) was added to the crude dissolved 50 mL acetone at
0 °C in small portions over a period of 1 h with vigorous stirring. The
mixture was stirred for another 1 h at the same temperature, then
diluted with CH2Cl2 (50 mL), filtered through paper filter and
evaporated to dryness. The residue was purified by column
chromatography on silica gel to give pure SiX as a yellow solid
(yield: 64%).
troduced as a pH-sensitive group. SiRB displayed the reversible H+
-
triggered ring-opening process of the corresponding spirocyclic struc-
tures accompanied by the remarkable chromogenic and fluorogenic
changes. Furthermore, SiRB probe showed the prominent fluorescence
changes in the pH range from 7.6 to 5.8, demonstrating an ideal suit-
ability for specific labeling of acidic microenvironment of tumor and
tracking the pH changes.
As a biodegradable polymer organic compound, PLGA (poly lactic-
co-glycolic acid) has good biocompatibility, non-toxicity, good en-
capsulation and film-forming properties. And it is widely used in
pharmaceutical, medical engineering materials and modern industry as
a pH-sensitive polymer material (Gu et al., 2015; Guimaraes et al.,
2015; Kanamala et al., 2016). In this work, a PLGA-based nanoparticle
is fabricated for ratiometric fluorescence imaging and pH detection. It is
found that two types of NIR dyes, SiRB and ICG (indocyanine green),
can be effectively encapsulated into PLGA via hydrophobic interaction
and induce the self-assembly of PLGA to form polymer-dye nano-
particles, with ICG, whose absorbance and fluorescence are inert to pH
change, serving as the internal reference item (Kim et al., 2015; Ruhm
et al., 2014). The pH-responsive dye SiRB could act as a pH indicator
under ratiometric fluorescence imaging. The accurate detection of
tumor pH by fluorescence imaging was realized after intravenous in-
jection of this nanoparticle. The gradual acidification of the tumor
microenvironment during the tumor growth, as well as the instant
tumor pH changes upon injection of external buffers, was vividly ob-
served by this method.
2.2.1.3. Synthesis of Br-B(2-(2′-bromophenyl)-6-butyl dioxazaborocan). N-
butyldiethanolamine (20 mmol) was added to
a suspension of 2-
bromophenylboronic acid (20 mmol) in anhydrous toluene (30 mL). The
mixture was heated at 50 °C for 2 h. After cooling to room temperature, the
toluene was evaporated under reduced pressure. The remaining clear
colorless crude oil was treated with heptane to remove the residual
toluene. The resulting suspension was allowed to stand at room
temperature overnight. The precipitated solid was collected by filtration,
washed with heptane, and dried overnight to give the title compound Br-B
as a white solid (yield: 58%).
2.2.1.4. Synthesis of SiRB. To a dried flask flushed with nitrogen,
compound Br-B (1.0 mmol) and anhydrous THF (10 mL) were added.
The solution was cooled to −78 °C, 1.6 M n-BuLi (n-Butyllithium)
(0.75 mL) was added over a period of 10 min and the mixture was
stirred for further 20 min. At the same temperature, a solution of SiX
(1.0 mmol) in anhydrous THF (10 mL) was slowly added. The mixture
was allowed to stir for 20 min at −78 °C, then allowed to warm to room
temperature gradually. After stirring for further 1 h, 6 M HCl solution
(10 mL) was added and the mixture was stirred for an additional
30 min. The resulting blue solution was neutralized with NaHCO3
solution, and extracted with CH2Cl2. The organic layer was washed
with brine and dried over anhydrous Na2SO4. After removal of the
solvent under reduced pressure, the residue was purified by column
chromatography on silica gel to give SiRB as a light blue solid (yield:
52%).
2. Materials and methods
2.1. Materials
3-Bromo-N,N-dimethylaniline, 2-bromophenylboronic acid (97%),
N-butyldi ethanolamine (98%) and indocyanine green (ICG) were
purchased from Aladdin. poly lactic-co-glycolic acid (PLGA, 99%) was
obtained from Fu Zhong Pharmaceutical Technology Co., Ltd (Beijing,
China). N-Butyl lithium, sec-butyllithium, heptane were obtained from
Sigma-Aldrich (St. Louis, Missouri, USA). Tetrahydrofuran (THF), me-
thylene chloride and acetic acid were purchased from Fuyu Fine
Chemical Co., Ltd (Tianjin, China). Other reagents were acquired from
Sigma-Aldrich. All the reagents were of analytical grade and used
without further purification.
2.2.2. Synthesis of PLGA-ICG-SiRB
The PLGA-ICG-SiRB nanocomposites were obtained by self-as-
sembly and subsequently loaded into PLGA nanoparticles. Briefly, 1 mg
synthesized SiRB, 0.2 mg ICG and 50 mg PLGA were firstly dispersed in
1 mL acetone. The mixture was added to 40 mg BSA (bovine serum
albumin) dispersed in 4 mL deionized water, and stirred overnight in
the dark. PLGA-ICG-SiRB nanoparticles were obtained after purification
of the suspensions by 8000–12,000 D dialysis bag.
2.2. Methods
2.2.1. Synthesis of SiRB
2.2.1.1. Synthesis of 4,4′-methlenebis (3-bromo-N,N-dimethylaniline)
(compound 1). A solution of 3-bromo-N,N-dimethylaniline (25 mmol)
in AcOH (80 mL) was added to 37% formaldehyde (125 mmol), and the
mixture was stirred at 85 °C for 90 min. After cooling to room
temperature, the reaction mixture was carefully neutralized with
saturated aqueous NaHCO3 and extracted with CH2Cl2 for three
times. The organic layer was washed with brine and dried over
2.2.3. Characterization of PLGA-ICG-SiRB
The PLGA-ICG-SiRB nanoparticles were characterized by transmis-
sion electron microscopy (TEM, Tecnai G2 20, FEI, USA). UV–vis ab-
sorption spectra were recorded using a UV–vis spectrometer (Lambda
35, Perkinelmer, USA). Fluorescence spectra were recorded with a RF-
33

Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
5301 fluorospectrophotometer (Shimadzu, Japan). The hydrodynamic
diameters of the obtained nanoparticles were determined by a dynamic
light scatter (DLS, Zetasizer Nano ZS-90, Malvern, England).
saline into the right shoulder of Kunming mice (18–22 g). When the
tumor reached a mean size of about 100 mm3 (~6 days after tumor
inoculation), the mice were intravenously injected with PLGA-ICG-SiRB
(200 μL per mouse). Fluorescence imaging of mice was performed at
preset times by in vivo imaging system (Bruker, Germany) equipped
with Kodak in Vivo Imaging System FX PRO (Carestream Health, Inc.,
USA). For fluorescence imaging, according to the UV–vis absorption
spectra obtained under 2.2.3 above, the entire tumor area was exposed
to the light (630 nm and 780 nm). The fluorescence images were cap-
tured at 650 nm and 830 nm emission wavelengths. The I650/I830
fluorescence signal ratios were calculated by the software and could be
used to measure the pH values in the tumor.
2.2.4. Cellular experiments of PLGA-ICG-SiRB
MCF-7 human breast cancer cell line was gained from Chinese
Academy of Sciences Cell Bank (Catalog No. HYC3204, Beijing, China).
Cells were cultured in DMEM with 10% FBS and 1% penicillin/strep-
tomycin in 5% CO2 and 95% air at 37 °C in a humidified incubator.
To assess the cancer cell toxicity of the PLGA-ICG-SiRB nano-
particles. MCF-7 cells were incubated with fresh medium containing
various concentrations of PLGA-ICG-SiRB, PLGA, ICG and SiRB for 24 h.
Finally, the cell viability assay was determined using SRB (sulforho-
damine B).
3. Results
2.2.5. Imaging experiment and pH detection
3.1. Synthesis and characterization of pH-sensitive NIR fluorescent probe
All animal experiments were performed under a protocol approved
by Henan laboratory animal center. The Life Science Ethics Committee
(Zhengzhou University) reviewed and approved the entire animal
protocol prior to initiation of the experiments. Mice tumor models were
generated by subcutaneous injection of 1 × 106 S180 cells in 0.1 mL
SiRB
SiRB was synthesized through a four-step method and its properties
were investigated. First, SiX was prepared by two-step reaction with 3-
bromo-N,N-dimethylaniline as a raw material. Then, the probe SiRB
Fig. 1. Synthesis and characterization of SiRB. (A) Synthesis of SiRB; (B) chemical structures of SiRB and the reversible transformation between boronate and boronic acid; (C) the color
changes of SiRB in different pH PBS solution (containing 1% DMSO); (D) UV–Vis absorbance spectra recorded in buffers with different pH values; (E) fluorescence spectra of SiRB
recorded in buffers with different pH values under 630 nm excitation; (F) the reversible fluorescence responses of SiRB (changes in the fluorescence at 647 nm between pH 7.6 and 5.8).
34

Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
was prepared by addition of a lithiated benzene moiety bearing a
protected boronic acid group to SiX, followed by deprotection in hy-
drochloric acid solution. In the protection of the boronic acid group, N-
butyl diethanolamine was chosen as a protective reagent because of its
tolerance to lithiation and effective deprotection (Fig. 1A) (Uno et al.,
2014). The structures of synthetic intermediates and SiRB were con-
firmed by 1HNMR spectroscopy (Fig. S1, Supporting information). The
H+-dependent color response of SiRB in PBS (phosphate buffer saline)
(containing 1% DMSO, dimethyl sulphoxide) was observed (Fig. 1B, C).
When the pH value of the solution was 7.4, the solution was colorless
and non-fluorescent. However, when the pH value was 5.8, the color of
the solution became light blue to give strong fluorescence.
To examine the spectral profile of SiRB, we measured the UV–Vis
absorption spectra and fluorescence emission spectra of SiRB in PBS at
different pH values. With the decrease of pH values range from 7.6 to
5.8, the simultaneous enhancements in UV–Vis absorption at 630 nm
and fluorescence intensity at 647 nm were noticed (Fig. 1D, E). The
UV–Vis absorption and fluorescence intensity of SiRB increased by 5.3
and 41 times range from pH 7.6 to 5.8, respectively. In order to further
study the mechanism of SiRB fluorescence switch, we conducted a
fluorescence switch reversibility experiment (Fig. 1F). After five cycles,
no noticeable changes in the emission were observed in SiRB. Mean-
while, the color of probe SiRB changed repeatedly between colorless
and blue.
PLGA-ICG-SiRB did not change obviously with the addition of salts at
pH 7.4 and 5.8 (Fig. 3B, C).
3.3. Cell experiment
We used breast cancer cell MCF-7 as a model cell for cell experi-
ments. It was essential the cytotoxicity of the prepared nanoparticle as
fluorescence imaging reagent. Thus, the cytotoxicity of PLGA-ICG-SiRB
in vitro was evaluated by the SRB method (Fig. 4A). Under the SiRB
concentration (20 μM), none of the four groups showed significant cy-
totoxicity. On the other side, when the concentration was higher than
20 μM, the cell viability declined slimly as the concentrations of PLGA-
ICG-SiRB, PLGA, ICG, SiRB increased. At the same concentration, the
cytotoxicity of ICG and SiRB was higher than that of PLGA and PLGA-
ICG-SiRB upon 40 μM, indicating that PLGA had almost no cytotoxicity
at normal dosing levels. The encapsulation of ICG and SiRB into PLGA
could effectively reduce the cytotoxicity of the probes.
From the laser confocal fluorescence microscopy experiment
(Fig. 4B–E), we have also surprisingly found that our synthetic fluor-
escent probe, SiRB, not only entered tumor cells, but also targeted ly-
sosomes in tumor cells. We stained MCF-7 cells with DAPI (4′,6-dia-
midino-2-phenylindole), LysoTracker Green DND-26 (commercially
available lysosome-specific probe) and SiRB. After 6 h staining, the
extra dyes would be washed and the cells were observed under laser
confocal fluorescence microscopy. DAPI could enter the nucleus and
emit blue fluorescence (Fig. 4B). LysoTracker Green DND-26 entered
the lysosome of the cell and releases green fluorescence (Fig. 4C). The
picture (Fig. 4D) showed the fluorescence emission of SiRB in cells and
Fig. 4E was the merged image. The experimental results showed that
the staining of probe SiRB fited well with that of LysoTracker Green
DND-26.
3.2. Synthesis and characterization of PLGA-ICG-SiRB
The PLGA-ICG-SiRB nanoparticle was synthesized and its properties
were investigated. We prepared the PLGA-ICG-SiRB by the method of
solvent emulsification and volatilization. The SiRB and ICG fluorescent
probes were encapsulated in PLGA polymers to form core-shell nano-
particle. The hydrodynamic diameter and apparent zeta potential of
PLGA-ICG-SiRB were measured by DLS (Figure, 2A, B). The average
3.4. Imaging experiment and pH detection
particle size of PLGA-ICG-SiRB was 95.13
zeta potential of PLGA-ICG-SiRB was −8.14
0.21 nm and the average
0.13 mV. We observed
Fluorescence imaging and pH detection of tumor was carried out by
recording the fluorescent signals of both SiRB and ICG from PLGA-ICG-
SiRB dispersed in serum solution with different pH values (Fig. 5A). The
fluorescent signals at 650 nm (emission peak of SiRB) exhibited an
obvious decrease as the increased pH value in the range of 5.8–7.6,
while the signals at 830 nm (emission peak of ICG) hardly changed
(Fig. 5B). The fluorescence intensity ratios between 650 nm and 830 nm
(650/830) also exhibited a sharp decrease in that range as the increase
of pH values (Fig. 5C). We performed linear regression calculations
based on the data in Fig. 5C. By calculation, we got the linear regression
equation: Y = 1.2X + 9.19, and the linear regression coefficient R2 was
0.9653. Due to the high linearity, it is shown that there is a linear re-
lationship between the fluorescence ratio and the pH value in the
measured pH range. We can use this linear regression equation to es-
timate the approximate pH values of tumor tissue and other major or-
gans.
We calculated the pH of tumor tissue and different organs by this
method. We constructed ascites tumor S180 into Kunming mice to
construct tumor-bearing mice animal model. To compare the differ-
ences in pH values between tumor and other major organs and illu-
minate the distribution of probes in different organs, ex vivo imaging of
major organs as well as tumors collected from mice at 6 h after i.v.
injection of PLGA-ICG-SiRB were conducted (Fig. 5D). As we can see
from Fig. 5E, the fluorescence intensity of the two probes at the tumor
site was significantly higher than that of other organs. The results of
Fig. 5F showed the relationship between the fluorescence ratio of the
two probes and the pH values. The data in the graphs were taken into
the linear regression equation to calculate the pH of the tumor site and
the value was 6.57. The pH of the remaining organs was approximately
7.45. We also calculated the changes in tumor pH values for different
growth days by this method (Fig. 5G). The results showed that with the
increase of the number of days of tumor growth, the fluorescence ratio
the morphology of the probe PLGA-ICG-SiRB by TEM (Fig. 2C). From
the figure of TEM, we can see the probes were packaged in PLGA to
form a core-shell structure and the whole preparation presented a
spherical morphology. Under pH 5.8, UV–Vis absorbance spectra of
different dye-loaded nanoparticles were measured (Fig. 2D), the results
suggested that PLGA-ICG-SiRB showed the characteristic peaks of both
ICG and SiRB were located at 780 nm and 630 nm, respectively. The
fluorescence spectra of ICG, PLGA-SiRB, PLGA-ICG and PLGA-SiRB-ICG
were also compared at pH 5.8 (Fig. 2E). As shown in Fig. 2F, the ab-
sorptions of ICG (780 nm) remained almost unchanged as the variation
of the pH values. However, the maximum UV–Vis absorptions of SiRB at
630 nm still significantly increased with decreasing pH. The ratio of the
maximum absorption of the two probes decreased with the increase of
pH values (Fig. 2G).
The time-dependent releases of SiRB and ICG from PLGA-ICG-SiRB
were measured across two pH values, 5.8 and 7.4 (Fig. 2H), which
represents the tumor acidic microenvironment and human normal
blood circulation pH value, respectively. As we can see from Fig. 2H,
the release rates of ICG and SiRB at pH 5.8 were significantly higher
than those at pH 7.4 in the serum solution in 60 h. In the acidic en-
vironment, the cumulative release rates of SiRB and ICG probes were
68% and 76%, respectively. However, in the neutral environment, the
cumulative release rates were only 9% and 11%.
The PLGA-ICG-SiRB nanoparticles also showed great stability in
different physiological solutions. First, we tested the stability of PLGA-
ICG-SiRB in serum solutions (pH 7.4). The results showed that the na-
noparticle in neutral serum solution behaved no significant change in
particle size in one week (Fig. 3A). To see if the salt substances in vivo
can affect the stability of PLGA-ICG-SiRB, we also added 20 mM NaCl,
KCl, CaCl2 to the serum solutions to imitate the human physiological
environment. The results showed that the UV–Vis absorption spectra of
35

Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
Fig. 2. Characterization of PLGA-SiRB-ICG. (A) and (B) DLS analysis results of PLGA-ICG-SiRB; (C) TEM images of PLGA-ICG-SiRB; (D) UV–Vis absorbance spectra of PLGA-ICG-SiRB;
PLGA-SiRB and PLGA-ICG; (E) fluorescence spectra of PLGA-SiRB, ICG, PLGA-ICG and PLGA-SiRB-ICG; (F) UV–Vis absorbance spectra of PLGA-ICG-SiRB recorded in buffer solutions at
different pH values; (G) the absorption ratio A630/A780 of PLGA-ICG-SiRB dispersed in buffers with different pH values; (H) The time-dependent release of SiRB and ICG from the PLGA-
ICG-SiRB nanoparticles in serum solution at pH 7.4 and 5.8.
Fig. 3. Stability of PLGA-ICG-SiRB nanoparticles. (A) DLS data of PLGA-ICG-SiRB dispersed in serum solution (pH 7.4) for different periods of time; (B) and (C) The absorption spectra of
PLGA-ICG-SiRB dispersed in pH 5.8 or 7.4 PBS (add 1% DMSO) with 20 mM NaCl, KCl or CaCl2.
36

Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
Fig. 4. Cellular experiment of the PLGA-ICG-SiRB nanoparticles. (A) Cell viability of MCF-7 cells at different concentrations of SiRB contained in the PLGA-ICG-SiRB nanoparticles after
24 h incubation; (B–E) Fluorescence images of MCF-7 cells incubated with PLGA-ICG-SiRB nanoparticles for 6 h (blue: cell nuclei stained by DAPI, green: lysosome stained by LysoTracker
Green DND-26, red: cell stained by PLGA-ICG-SiRB). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
was also increasing, and the pH value of tumor microenvironment was
decreasing. The pH values of tumor site from 7 days to 19 days were
6.83, 6.60, 6.38, 6.19 and 6.01, respectively. This finding has the po-
tential to be used in defining tumor stages and detecting tumor pro-
gression.
blue color change in acidic environment. These phenomena implied
that the boron-containing probe SiRB was sensitive to H+, and the re-
versible color change was likely to be induced by the reversible ring-
opening process of the corresponding rings (Lukinavicius et al., 2013).
Based on the probe SiRB, we prepared the PLGA-ICG-SiRB nano-
particle which can be used to accurately reflect the pH value of tumor
and other organs by fluorescence ratio imaging. The ring opening
process of the borate-based spiro-rings in the SiRB dye as the decrease
of pH, would lead to enhance the absorption of SiRB, as well as in-
creased fluorescence emission. On the other hand, the fluorescence of
ICG, different from that of SiRB, is inert to pH changes. So ICG may
serve as the internal reference when SiRB is used for pH sensing.
4. Discussion
In this paper, we successfully synthesized and characterized the
near-infrared fluorescence probe SiRB, which has a sensitive response
at pH 5.8–7.6. SiRB formed a colorless solution and no fluorescence was
emitted in a neutral buffer solution, but underwent a reversible light
37

Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
Fig. 5. The pH-responsive fluorescence imaging. (A) Fluorescence imaging of PLGA-ICG-SiRB dispersed in serum solution with different pH values; (B) fluorescence intensities of ICG and
SiRB under different pH values; (C) I650/I830 intensity ratio of PLGA-ICG-SiRB measured under different pH values; (D) ex vivo fluorescence images of major organs and tumor dissected
from mice i.v. injected with PLGA-ICG-SiRB (24 h post-injection). Li: liver, Sp: spleen, Ki: kidney, H: heart, Lu: lung, T: tumor; (E) fluorescence intensity at 650 nm and 830 nm; (F) the
I650/I830 intensity ratio in the tumor as well as other major organs measured in (D); (G) ex vivo fluorescence images of tumor with different sizes collected from mice i.v. injected with
PLGA-ICG-SiRB (24 h post-injection); (H) fluorescence intensity at 650 nm and 830 nm; (I) the I650/I830 intensity ratio in the tumors measured in (G).
Therefore, the PLGA-ICG-SiRB showed strong pH-dependent absor-
bance. The TEM images showed that PLGA encapsulated two kinds of
probes to form a core-shell structure of nanoparticles. The UV absorp-
tion spectra also showed that PLGA-ICG-SiRB had two characteristic
absorption peaks of 630 nm (the peak of SiRB) and 780 nm (the peak of
ICG), respectively. This result further demonstrated that both probes
were successfully packed in the PLGA.
In vitro cytotoxicity studies, the cell viability of the preparation
group was significantly higher than that of the free probe group, in-
dicating that the encapsulation of PLGA reduced the cytotoxicity of the
probe. Moreover, the PLGA-ICG-SiRB was still non-toxic to cells even at
higher probe concentrations, which was attributed to the excellent
biocompatibility of PLGA (de Groot et al., 2017). The probe release
experimental results in vitro showed that PLGA can split itself in re-
sponse to the slightly acidic environment, which provided the basis for
the specific release of the probes (Kang et al., 2017; Santhosh et al.,
2017).
not change significantly within the measured time range, indicating
that the stability of the nanoparticles was superior. The addition of
NaCl, KCl, CaCl2 to the buffer solution did not affect the UV absorption
of the PLGA-ICG-SiRB, indicating that the salts did not affect the
structure of the nanoparticles and the fluorescence emission properties.
Moreover, we measured their UV absorbance at different pH solution,
indicating that these salts did not affect the fluorescence switch func-
tion of PLGA-ICG-SiRB.
In the laser confocal fluorescence microscope, we were surprised to
find that the fluorescent probe SiRB has a good ability to enter the cell
and certain lysosomal targeting effect. These results demonstrated that
probe SiRB possessed great cell membrane permeability and suitability
of specific labeling of acidic lysosomes in living cells. In living cells,
lysosome is a major subcellular organelle that contains numerous en-
zymes and protein displaying a variety of activities and function at pH
values (4.5–5.5). Searching for lysosome-targetable fluorescent probes
is an active field as well as a challenge for the analytical chemistry
research effort (Liang et al., 2018). So the probe we prepared is pro-
mising for various studies targeting lysosomes. We speculate the effi-
cient lysosome-targeting location of SiRB without the intentionally
In this paper, we also examined the stability of PLGA-ICG-SiRB and
the effects of common salts on the fluorescence emission. The experi-
mental results showed that the particle size of the PLGA-ICG-SiRB did
38

Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
functionalized lysosome-locating group was attributed to the H+-trig-
gered ring-opening process with appropriate pKcycl value that matched
well with the pH value of the acidic lysosomes.
marker Snail and the prognostic impact of lymphocytes in the tumor microenviron-
ment in invasive ductal breast cancer. Exp. Mol. Pathol. 102, 268–275.
Chen, J., Li, Y., Lv, K., Zhong, W., Wang, H., Wu, Z., 2016. Cyclam-functionalized carbon
dots sensor for sensitive and selective detection of copper (II) ion and sulfide anion in
aqueous media and its imaging in live cells. Sensors Actuators B Chem. 224, 298–306.
de Groot, A.M., Du, G., Mönkäre, J., Platteel, A.C., Broere, F., Bouwstra, J.A., 2017.
Hollow microneedle-mediated intradermal delivery of model vaccine antigen-loaded
PLGA nanoparticles elicits protective T cell-mediated immunity to an intracellular
bacterium. J. Control. Release 266, 27–35.
Dervisevic, M., Senel, M., Cevik, E., 2017. Novel impedimetric dopamine biosensor based
on boronic acid functional polythiophene modified electrodes. Mater. Sci. Eng., C 72,
641–649.
Florence, M.E., Massuda, J.Y., Soares, T.C., Stelini, R.F., Poppe, L.M., Brocker, E.B., de
Souza EM, 2015. p53 immunoexpression in stepwise progression of cutaneous
squamous cell carcinoma and correlation with angiogenesis and cellular prolifera-
tion. Pathol. Res. Pract. 211, 782–788.
Gandellini, P., Andriani, F., Merlino, G., D'Aiuto, F., Roz, L., Callari, M., 2015. Complexity
in the tumour microenvironment: Cancer associated fibroblast gene expression pat-
terns identify both common and unique features of tumour-stroma crosstalk across
cancer types. Semin. Cancer Biol. 35, 96–106.
Gu, B., Wang, Y., Burgess, D.J., 2015. In vitro and in vivo performance of dexamethasone
loaded PLGA microspheres prepared using polymer blends. Int. J. Pharm. 496,
534–540.
Guimaraes, P.P., Oliveira, M.F., Gomes, A.D., Gontijo, S.M., Cortes, M.E., 2015. PLGA
nanofibers improve the antitumoral effect of daunorubicin. Biointerfaces 136,
248–255.
H.S, G., Melavanki, R.M., D, N., P, B., Kusanur, R.A., 2017. Binding of boronic acids with
sugars in aqueous solution at physiological pH - estimation of association and dis-
sociation constants using spectroscopic method. J. Mol. Liq. 227, 37–43.
Hui, L., Chen, Y., 2015. Tumor microenvironment: sanctuary of the devil. Cancer Lett.
368, 7–13.
Joyce, J.A., 2005. Therapeutic targeting of the tumor microenvironment. Cancer cell 7,
513–520.
Kanamala, M., Wilson, W.R., Yang, M., Palmer, B.D., Wu, Z., 2016. Mechanisms and
biomaterials in pH-responsive tumour targeted drug delivery: a review. Biomaterials
85, 152–167.
Kang, B.S., Choi, J.S., Lee, S.E., Lee, J.K., Kim, T.H., Jang, W.S., 2017. Enhancing the in
vitro anticancer activity of albendazole incorporated into chitosan-coated PLGA na-
noparticles. Carbohydr. Polym. 159, 39–47.
Kim, T.I., Jeong, K.H., Shin, M.K., 2015. Verrucous epidermal nevus (VEN) successfully
treated with indocyanine green (ICG) photodynamic therapy (PDT). JAAD Case Rep.
1, 312–314.
Lau, J.T., Lo, P.C., Jiang, X.J., Wang, Q., Ng, D.K., 2014. A dual activatable photo-
sensitizer toward targeted photodynamic therapy. J. Med. Chem. 57, 4088–4097.
Li, B., Gb, Ge, Wen, L., Yuan, Y., Zhang, R., Peng, X., 2017. A lysosomal probe for
monitoring of pH in living cells and ovarian tumour. Dyes Pigments 139, 318–325.
Liang, B., Wang, B., Ma, Q., Xie, C., Li, X., Wang, S., 2018. A lysosome-targetable turn-on
fluorescent probe for the detection of thiols in living cells based on a 1,8-naphtha-
limide derivative. Spectrochim. Acta, Part A 192, 67–74.
Lukinavicius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., 2013. A near-in-
frared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat.
Chem. 5, 132–139.
Robert, J.G., Ronald, M.L., 2001. Frontiers in the measurement of cell and tissue pH.
Novartis Found. Symp. 240, 7–22.
Ruhm, A., Gobel, W., Sroka, R., Stepp, H., 2014. ICG-assisted blood vessel detection
during stereotactic neurosurgery: simulation study on excitation power limitations
due to thermal effects in human brain tissue. Photodiagn. Photodyn. Ther. 11,
307–318.
In the vitro fluorescence imaging experiment, we calculated the
linear regression equation between the fluorescence ratio and the pH
value by calculating the ratio of the two fluorescence intensities at
different pH values. Since the linear regression equation has a good
linearity coefficient R2, we can calculate the corresponding pH value by
substituting the fluorescence emission data into the equation. Robert
and Ronald reported the methods that using magnetic resonance
spectroscopy (MRS), pH-sensitive proteins and pH-sensitive MR con-
trast agents to measure cell and tissue pH (Robert and Ronald, 2001).
We estimated the pH of the tumor site, the results were consistent with
that obtained by using the method of Robert's.
Due to the auto-fluorescence of the animal and the limited pene-
tration ability of fluorescent imaging, we first dissected the various
organs of the mice, and then carried out fluorescence imaging experi-
ments. The results showed that the pH value of the tumor site was
significantly lower than that of other organs, and the concentration of
nanoparticles in the tumor site was significantly higher than that of
other organs. Because the fluorescence intensity of ICG does not change
with the pH value, while relates with its concentration, it can be shown
that PLGA-ICG-SiRB in the tumor site aggregation. The rather efficient
tumor homing of the nanoparticles may be due to the enhanced per-
meability and retention effect. Then, we detected the pH value of the
tumor with different growth days. The experimental results showed that
with the growth of the tumor, the tumor microenvironment acidity will
continue to increase in the measured range. This may be due to the
continuous accumulation of acidic substances during the growth of the
tumor leading to a decrease in the pH value of the tumor micro-
environment. Based on above results, the PLGA-ICG-SiRB pH detection
system will exert potential theranostic effect in future tumor diagnosis
and treatment.
5. Conclusions
In conclusion, we successfully synthesized the near-infrared fluor-
escence probe SiRB, which has a sensitive response at pH 5.8–7.6. Based
on this probe, we can prepare the PLGA-ICG-SiRB nanoparticles which
can be used to accurately reflect the pH value of tumor by fluorescence
ratio imaging. The PLGA-ICG-SiRB nanoparticles have dual pH sensi-
tivity. So, after the accumulation in the tumor by EPR effect, the PLGA
accelerates the cleavage to release the probes in the tumor acidic en-
vironment. SiRB is excited by H+ to transform from the quenched state
to the excited state. By the fluorescence intensity ratio changes of the
PLGA-ICG-SiRB probe can accurately reflect the tumor pH values and
pH changes. The applications of the PLGA-ICG-SiRB probe in tumor
detection and the research on tumor microenvironment have great
potential.
Santhosh, K.T., Alizadeh, A., Karimi-Abdolrezaee, S., 2017. Design and optimization of
PLGA microparticles for controlled and local delivery of Neuregulin-1 in traumatic
spinal cord injury. J. Control. Release 261, 147–162.
Tao, J., Wang, X.C., Chen, X.Q., Li, T.C., Diao, Q.P., Yu, H.F., 2017. Si-rhodamine B
thiolactone: a simple long wavelength operating chemosensor for direct visual re-
cognition of Hg2+. Dyes Pigments 137, 601–607.
Uno, S.N., Kamiya, M., Yoshihara, T., Sugawara, K., Okabe, K., Tarhan, M.C., 2014. A
spontaneously blinking fluorophore based on intramolecular spirocyclization for live-
cell super-resolution imaging. Nat. Chem. 6, 681–689.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ejps.2018.03.015.
Wang, T., Zhao, Q.J., Hu, H.G., Yu, S.C., Liu, X., Liu, L., 2012. Spirolactonized Si-rho-
damine: a novel NIR fluorophore utilized as a platform to construct Si-rhodamine-
based probes. Chem. Commun. 48, 8781–8783.
Acknowledgement
Wang, H., Zhang, P., Hong, Y., Zhao, B., Yi, P., Chen, J., 2017. Ratiometric imaging of
lysosomal hypochlorous acid enabled by FRET-based polymer dots. Polym. Chem. 8,
5795–5802.
The authors gratefully acknowledge financial support from the
Scientific and Technological Project of Henan Province of China (Grant
no. 52110131).
Wu, T., Dai, Y., 2017. Tumor microenvironment and therapeutic response. Cancer Lett.
387, 61–68.
Yan, M., Jurasz, P., 2016. The role of platelets in the tumor microenvironment: from solid
tumors to leukemia. Biochim. Biophys. Acta 1863, 392–400.
Yang, F., Yu, Y., 2015. Tumor microenvironment-the critical element of tumor metastasis.
Chin. J. Lung Cancer 18, 48–54.
Zhao, Y., Ren, W., Zhong, T., Zhang, S., Huang, D., Guo, Y., 2016. Tumor-specific pH-
responsive peptide-modified pH-sensitive liposomes containing doxorubicin for en-
hancing glioma targeting and anti-tumor activity. J. Control. Release 222, 56–66.
Zhu, W.W., Chai, X.Y., Wang, B.G., Zou, Y., Wang, T., Meng, Q.G., 2015. Spiroboronate
Si-rhodamine as a near-infrared probe for imaging lysosomes based on the reversible
ring-opening process. Chem. Commun. 51, 9608–9611.
Disclosure
The authors declare no competing financial interest.
References
Alkatout, I., Hubner, F., Wenners, A., Hedderich, J., Wiedermann, M., Sanchez, 2017. In
situ localization of tumor cells associated with the epithelial-mesenchymal transition
39